Rapid advances in biotechnology have revolutionized the production of medically beneficial bioactive molecules. We aim in this chapter to provide an overview of the role of biotechnology in the synthesis and enhancement of bioactive compounds. Polyphenols, flavonoids, alkaloids, and terpenoids have powerful antioxidant, anti-inflammatory, antimicrobial, and anticancer properties. Low yield, environmental impact, and sustainability challenges often accompany traditional extraction methods. Through genetic engineering, metabolic engineering, and synthetic biology, biotechnology offers innovative solutions. Using genetic engineering techniques, such as gene cloning and expression, desired bioactive compounds can be produced in large quantities in microbial hosts. Through metabolic engineering, these production pathways are further optimized by altering the metabolic networks within the host organisms. The field of synthetic biology uses engineering principles to design and construct novel biological parts, devices, and systems, thus improving bioactive molecule production. Plant biotechnology is extensively investigated for the production of bioactive molecules. In addition, a number of different techniques, such as tissue culture, genetic alteration, or elicitation, are also used in this procedure. Biotechnological approaches have the potential to discover and produce novel bioactive compounds, ultimately improving human well-being and healthcare.